Steroid receptor coactivator-3 (SRC-3) is the second most overexpressed oncogene that plays a significant role in the development of breast cancer. Despite its importance as an oncogene, whether and how SRC-3 promotes cancer stem cells (CSCs) activity to drive aggressive cancer development remains unknown. In this application, we address the challenge of understanding how SRC-3 drives aggressive breast cancer development, and uncover a novel and unexpected mechanism for the function of SRC-3 in CSCs. Interestingly, SRC-3 functions as a corepressor and not a coactivator to suppress expression of miR-34a, a critical suppressor of CSCs' activity. Expression of SRC-3 is positively correlated with tumor grades and stages, but inversely correlated with expression of miR-34a in breast cancer, suggesting a causal relationship between SRC-3 and miR-34a expression and disease state. Our study identifies the first corepressor activity of SRC-3, and SRC-3 as the first suppressor of miR-34a expression in CSCs. Our hypothesis is that SRC-3 functions as a corepressor in a context- and signaling-dependent manner to promote CSCs activity. The objective of this application is to identify the mechanism which dictates the corepressor function of SRC-3, and to elucidate the significance of this corepressor function in the progression of breast cancer. We propose three aims to achieve our objective. Aim 1. Elucidate the mechanism for the unexpected corepressor function of SRC-3. To understand how miR-34a is suppressed, transcription repressor regulatory factor X1 (RFX1) and de-phosphorylation of SRC-3 at S505 were identified as potential determinants for the suppression of miR-34a in CSCs. Our objective is to elucidate how de-phosphorylation of S505 dictates the suppression of miR-34a by SRC-3 and RFX1 in CSCs. Aim 2. Determine the function of SRC-3 S505 de-phosphorylation in cancer stem cells. SRC-3 and RFX1 are identified to be novel suppressors of miR-34a, and importantly, suppression of miR-34a requires de-phosphorylation of SRC-3 S505. The objective of this aim is to demonstrate that de-phosphorylation of S505 plays an important role in the ability of SRC-3/RFX1 to promote CSCs function. Aim 3. Elucidate the functional importance of HSP70 K246 deacetylation. CSCs play an important role in therapy resistance. Elucidating the therapy resistant mechanism of SRC-3-miR-34a CSCs pathway is clearly important. We identified HDAC1 and HDAC7 as novel targets of miR-34a, and showed that HDACs1 and 7 deacetylate HSP70 at K246. Our objective is to demonstrate that HDACs1 and 7 promote the therapy resistant activity of SRC-3-miR-34a CSCs pathway by deacetylating HSP70 K246, and to identify the resistant mechanism of K246 deacetylation. Our study is innovative. It uncovers a novel corepressor activity that governs the function of oncogenic coactivator SRC-3 in CSCs, and reveals an important role of transcriptional repressor RFX1 and de-phosphorylation of S505. Our study is expected to have a significant impact on the diagnosis and development of targeted cancer therapy to treat aggressive and therapy resistant breast cancer.